Interferon-β (IFN-β) stabilizes the blood–brain barrier (BBB) in vitro. Here we investigated the effect of serum from 15 IFN-β-1b-treated multiple sclerosis (MS) patients on the permeability read-outs of small solutes in an in vitro BBB model consisting of human brain microvascular endothelial cells in co-culture with rat astrocytes. The addition of sera from IFN-β-treated patients resulted in a significantly (p < 0.05) reduced permeability as compared with untreated patients. Our findings show that sera from IFN-β-1b-treated MS patients have a stabilizing effect on the in vitro BBB. We suggest an unknown potentially pro-inflammatory factor in the serum of MS patients that may lead to a BBB dysfunction and can be modulated by IFN-β.